Treatment of cholera

ABSTRACT

This invention relates to treatment of cholera and related conditions using oligosaccharide compositions which bind V. cholerae toxin and/or one or more serotypes of the organism V. cholerae. More specifically, the invention concerns neutralization and removal of V. cholerae toxin and/or organisms from the intestinal tract.

FIELD OF THE INVENTION

This invention relates to treatment of cholera. More specifically, theinvention concerns neutralization and elimination of cholera toxin. Thisinvention also relates to binding and removal of Vibrio cholerae, thecausative agent of cholera from the intestinal tract.

REFERENCES

The following references are cited in the application as numbers inbrackets ([ ]) at the relevant portion of the application.

1. Merritt, Ethan A., et al., "Crystal structure of cholera toxinB-pentamer bound to receptor G_(M1) pentasaccharide", Protein Science,3: 166-175 (1994).

2. Spangler, Brenda D., "Structure and Function of Cholera Toxin and theRelated Escherichia coli Heat-Labile Enterotoxin", MicrobiologicalReviews, 56, No. 4:622-647 (1992).

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4. Fishman, Peter H., et al., "Gangliosides as Receptors for BacterialEnterotoxins", Advances in Lipid Research, 25:165-187 (1993).

5. Lanne et al., "On the role of the carboxyl group of sialic acid inbinding of cholera toxin to the receptor glycosphingolipid, GM1", J.Biochem., 116: 1269-1274 (1994).

6. Schengrund et al., "Binding of Vibrio cholera toxin and heat-labileenterotoxin of Escherichia coli to GM1, derivatives of GM1 and nonlipidoligosaccharide polyvalent ligands", J. Biol. Chem., 264:13233-13237(1989).

7. Fukuda et al., "Comparison of the carbohydrate-binding specificitiesof cholera toxin and Escherichia coil heat-labile enterotoxins LTh-I,LTh-IIa, and LTh-IIb", Infect. Immun., 56: 1748-1753(1988).

8. Uesaka et al., "Simple method of purification of Escherichia coliheat-labile enterotoxin and cholera toxin using immobilized galactose",Microb. Path., 16:71-76 (1994).

9. Tayot et al., "Receptor-specific large-scale purification of choleratoxin on silica beads derivatized with lysoGM1 ganglioside", Eur. J.Biochem. 113: 249-58 (1981).

10. Parikh et al., "Ganglioside-agarose and cholera toxin", Meth.Enzymol., 34:610-619(1974).

11. Lemieux, R. U., et al., "The properties of a `synthetic` antigenrelated to the blood-group Lewis A", J. Am. Chem. Soc., 97:4076-83(1975).

12. Lemieux, R. U., et al., "Glycoside-Ether-Ester Compounds", U.S. Pat.No. 4,137,401, issued Jan. 30, 1979.

13. Lemieux, R. U., et al., "Artificial Oligosaccharide AntigenicDeterminants", U.S. Pat. No. 4,238,473, issued Dec. 9, 1980.

14. Lemieux, R. U., et al., "Synthesis of 2-Amino-2-Deoxyglycoses and2-Amino-2-Deoxyglycosides from glycals", U.S. Pat. No. 4,362,720, issuedDec. 7, 1982.

15. Cox, D., et al. "A New Synthesis of4-O-α-D-Galactopyranosyl-D-Galacto-Pyranose", Carbohy. Res., 62:245-252(1978).

16. Dahmen, J., et al., "Synthesis of space arm, lipid, and ethylglycosides of the trisaccharide portion[α-D-Gal-(1-4)-β-D-Gal(1-4)-β-D-Glc] of the blood group p^(k) antigen:preparation of neoglycoproteins", Carbohydrate Research, 127:15-25(1984).

17. Garegg, P. J., et al., "A Synthesis of 8-Methoxycarbonyloct-1-ylO-α-D-Galactopyranosyl-(1-3)-O-β-D-Galactopyranosyl-(1-4)-2-Acetamido-2-Deoxy-β-D-Glucopyranoside",Carbohy. Res., 136:207-213 (1985).

18. Garegg, P. J., et al., "Synthesis of 6- and 6'-deoxy derivatives ofmethyl 4-O-α-D-galactopyranosyl-β-D-galactopyranoside for studies ofinhibition of pyelonephritogenic fimbriated E. coli adhesion to urinaryepithelium-cell surfaces", Carbohy. Res., 137:270-275 (1985).

19. Jacquinet, J. C., et al., "Synthesis of Blood-group Substances, Part11. Synthesis of the TrisaccharideO-α-D-Galactopyranosyl-(1-3)-O-β-D-galactopyranosyl-(1-4)-2-acetamido-2-deoxy-D-glucopyranose",J. C. S. Perkin, I: 326-330 (1981).

20. Koike, K., et al., "Total Synthesis of Globotriaosyl-E andZ-Ceramides and Isoglobotriaosyl-E-Ceramide," Carbohydr. Res., 163:189-208 (1987).

21. Schaubach, R., et al., "Tumor-Associated Antigen Synthesis:Synthesis of the Gal-α-(1-3)-Gal-β-(1-4)-GlcNAc Epitope. A specificDeterminant for Metastatic Progression?", Liebigs Ann. Chem., 607-614(1991).

22. Ratcliffe, R. M., et al., "Sialic Acid Glycosides, Antigens,Immunoadsorbents, and Methods for Their Preparation", U.S. Pat. No.5,079,353, issued Jan. 7, 1992.

23. Okamoto, K., et al., "Glycosidation of Sialic Acid," Tetrahedron,47:5835-5857 (1990).

24. Abbas, S. A., et al., "Tumor-Associated Oligosaccharides I:Synthesis of Sialyl-Lewis^(a) Antigenic Determinant", Sialic Acids,Proc. Japan-German Symp. Berlin 22-23 (1988).

25. Paulsen, "Advances in Selective Chemical Syntheses of ComplexOligosaccharides", Angew. Chem. Int. Ed. Eng., 21:155-173 (1982).

26. Schmidt, "New Methods for the Synthesis of Glycosides andOligosaccharides--Are There Alternatives to the Koenigs-Knorr Method?",Angew. Chem. Int. Ed. Eng., 25:212-235 (1986).

27. Fugedi, P., et al., "Thioglycosides as Glycosylating Agents inOligosaccharide Synthesis", Glycoconjugate J., 4:97-108 (1987).

28. Kameyama, A., et al., "Total synthesis of sialyl Lewis X",Carbohydrate Res., 209:c1-c4 (1991).

29. Ekborg, G., et al., "Synthesis of Three Disaccharides for thePreparation of Immunogens bearing Immunodeterminants Known to Occur onGlycoproteins", Carbohydrate Research, 110:55-67 (1982).

30. Dahmen, J., et al., "2-Bromoethyl glycosides: applications in thesynthesis of spacer-arm glycosides", Carbohydrate Research, 118:292-301(1983).

31. Rana, S. S., et al., "Synthesis of Phenyl2-Acetamido-2-Deoxy-3-O-α-L-Fucopyranosyl-β-D-Glucopyranoside andRelated Compounds", Carbohydrate Research, 91:149-157 (1981).

32. Amvam-Zollo, P., et al., "Streptococcus pneumoniae Type XIVPolysaccharide: Synthesis of a Repeating Branched Tetrasaccharide withDioxa-Type Spacer-Arms", Carbohydrate Research, 150:199-212 (1986).

33. Paulsen, H., "Synthese von oligosaccharid-determinanten mitmid-spacer vom typ des T-antigens", Carbohydr. Res., 104:195-219 (1982).

34. Chernyak, A. Y., et al., "A New Type of Carbohydrate-ContainingSynthetic Antigen: Synthesis of Carbohydrate-Containing PolyacrylamideCopolymers having the Specificity of 0:3 and 0:4 Factors of Salmonella",Carbohydrate Research, 128:269-282 (1984).

35. Fernandez-Santana, V., et al., "Glycosides of Monoallyl DiethyleneGlycol. A New type of Spacer group for Synthetic Oligosaccharides", J.Carbohydrate Chemistry, 8(3): 531-537 (1989).

36. Lee, R. T., et al., "Synthesis of 3-(2-Aminoethylthio)PropylGlycosides", Carbohydrate Research, 37:193-201 (1974).

37. Armstrong, G. D., et al., "Investigation of shiga-like toxin bindingto chemically synthesized oligosaccharide sequences", J. Infect. Dis.,164:1160-1167 (1991).

38. Heerze, L. D. et al., "Oligosaccharide sequences attached to aninert support(SYNSORB) as potential therapy for antibiotic-associateddiarrhea and pseudomembranous colitis", J. Infect. Dis., 169:1291-1296(1994).

39. U.S. patent application Ser. No. 08/195,009, filed Feb. 14, 1994, byHeerze, et al., for TREATMENT OF ANTIBIOTIC ASSOCIATED DIARRHEA(allowed).

40. U.S. patent application Ser. No. 08/126,645, filed Sep. 27, 1993 byArmstrong, et al., for DIAGNOSIS AND TREATMENT OF BACTERIAL DYSENTERY.

41. U.S. patent application Ser. No. 07/996,913, filed Dec. 28, 1992, byArmstrong, for DIAGNOSIS AND TREATMENT OF BACTERIAL DYSENTERY.

The disclosure of the above publications, patents and patentapplications are herein incorporated by reference in their entirety tothe same extent as if the language of each individual publication,patent and patent application were specifically and individuallyincluded herein.

BACKGROUND OF THE INVENTION

Cholera is a severe diarrheal disease that affects approximately 3million individuals per year worldwide (mainly in less developedcountries). It is caused by consuming food or drinking watercontaminated with the microorganism Vibrio cholerae. When the organismis ingested, it has the ability to colonize the intestinal tract.

In the small intestine, V. cholerae attaches to the intestinal mucosaand releases exotoxins, the most important being cholera toxin (CT),which act on mucosal cells [1-4]. The action of CT on intestinal cellsinduces fluid secretion and increased permeability of electrolytes intothe small intestine resulting in severe diarrhea and electrolyteimbalance.

Two other toxins are also produced by V. cholerae. They include zonaoccludens toxin (Zot) which disrupts tight junctions between cells, andaccessory cholera enterotoxin (Ace), which causes diarrhea in animals.The role of these two toxins in the overall pathogenesis of the diseaseremains unclear.

An additional cytolytic toxin is produced by O1 E1 Tor and O139serotypes of V. cholerae. This toxin has a hemolytic and cytotoxicactivity which appears to play a role in the pathogenesis of cholera.

Mortality rates are high for infants and children that are inflictedwith cholera. The current method of treatment for cholera is to replacefluids and restore electrolyte balance.

Not all strains of V. cholerae are responsible for causing disease. Thedisease causing strains belong to the O1 serotype which includes theclassical and the E1 Tor biotypes. All other serotypes except for oneare thought to be nonvirulent or capable of causing only minor diarrhea.The only non O1 strain of V. cholerae that has been shown to causefull-blown cholera was identified two years ago. It belongs to the O139serotype. This serotype has been identified as the causal agent forrecent outbreaks of cholera in Asia. It produces all the virulencefactors (including CT) associated with the O1 serotypes of V. cholerae.

The virulence factors most important for causing disease are the toxincoregulated pili (Tcp) which allow V. cholerae to colonize the smallintestine. Although the host cell receptor has yet to be identified forpili, there is some indirect evidence which suggests that a carbohydratemay be involved. This evidence is based on the finding that individualswho have the O blood group are more susceptible to severe cases ofcholera while people who are AB blood group positive tend to be somewhatresistant toward the disease. One possible explanation for this findingis that the pili found on V. cholerae may use the O blood groupoligosaccharide structure for colonization of the small intestine, thusrendering individuals with the O blood group more susceptible todisease.

CT is the virulence factor most responsible for the symptoms of thedisease. CT possesses an enzymatic activity which elevates the levels ofcyclic AMP (cAMP) in host cells. The increase in cAMP levels alters theion transport systems within cells thus affecting the osmotic balancewithin the intestine that leads to diarrhea. CT utilizes the gangliosideGM1 (βGal(1-3)βGalNAc(1-4)[αNeuAc(2-3)]βGal(1-4)βGlc-ceramide) to bindto host cell receptors.

Cholera toxin (CT) has been shown to bind to several derivatives of theganglioside GM1 where the carboxyl group of sialic acid had beenmodified to form a number of C(1) amides [5]. The structure of thesecompounds is: βGal(1-3)βGalNAc(1-4)[αNeuAcR(2-3)]βGal(1-4)βGlc-ceramide,where R is selected from the group consisting of amide, methylamide,ethylamide, propylamide, and benzylamide of sialic acid.

Other derivatives of GM1 that were shown to bind CT include [6]:βGal(1-3)βGalNH2(1-4)[αNeu-NH2(2-3)]βGal(1-4)βGlc-ceramide;βGal(1-3)βGalNAc(1-4)[αNeuAcR(2-3)]βGal(1-4)βGlc-ceramide, where R isthe methyl ester of sialic acid;βGal(1-3)βGalNAc(1-4)[α(C7)NeuAc(2-3)]βGal(1-4)βGlc-ceramide; andβGal(1-3)βGalNAc(1-4)[αNeuAcR(2-3)]βGal(1-4)βGlc-ceramide, where R isethanolamineamide.

Other gangliosides which have been shown to bind CT include [6,7]: GM2(βGalNAc(1-4)[αNeuAc(2-3)]βGal(1-4)βGlc-ceramide) and GD1b(βGal(1-3)βGalNAc(1-4)[αNeuAc(2-3)αNeuAc(2-3)]βGal(1-4)βGlc-ceramide.

In addition, highly purified CT preparations have been obtained usinglyso GM1 ganglioside or galactose affinity columns [8-10].

With respect to methods of diagnosis of the presence of CT in a sample,one method for detecting Vibrio cholerae in a sample is to culture thesample. The disadvantages of this method include the length of timerequired and interference by non-pathogenic, i.e., non-toxin producing,V. cholerae strains. Other methods involve the use of specific antiseraor monoclonal antibodies.

In view of the above, there is a need for a compound which would treatcholera. A preferred compound would be administered noninvasively, suchas orally, and would specifically remove toxin and/or organisms from theintestinal tract.

SUMMARY OF THE INVENTION

The invention provides compositions and methods for the treatment ofcholera and associated symptoms caused by cholera toxin.

The invention also provides compositions and methods for the treatmentof cholera and associated symptoms caused by colonization of thegastrointestinal tract by V. cholerae.

In one aspect, the invention provides a method to treat cholera in asubject, which method comprises administering to a subject in need ofsuch treatment an effective amount of a composition comprising anoligosaccharide sequence covalently attached to a pharmaceuticallyacceptable solid, inert support through a non-peptidyl compatible linkerarm, wherein said oligosaccharide sequence binds cholera toxin, andwherein said composition is capable of being eliminated from thegastrointestinal tract.

In a further aspect, the invention provides a pharmaceutical compositionuseful in treating cholera and related conditions initiated by choleratoxin, which composition comprises an oligosaccharide sequencecovalently attached to a pharmaceutically acceptable solid, inertsupport through a non-peptidyl compatible linker arm, wherein saidoligosaccharide sequence binds cholera toxin; and a pharmaceuticallyacceptable carrier, wherein said composition is capable of beingeliminated from the gastrointestinal tract.

In yet a further aspect, the invention provides a method to treatcholera in a subject, which method comprises administering to a subjectin need of such treatment an effective amount of a compositioncomprising an oligosaccharide sequence covalently attached to apharmaceutically acceptable solid, inert support through a non-peptidylcompatible linker arm, wherein said oligosaccharide sequence binds V.cholerae, and wherein said composition is capable of binding themicroorganism so that it is eliminated from the gastrointestinal tract.

In a still further aspect, the invention provides a pharmaceuticalcomposition useful in treating cholera and related conditions, whichcomposition comprises an oligosaccharide sequence covalently attached toa pharmaceutically acceptable solid, inert support through anon-peptidyl compatible linker arm, wherein said oligosaccharidesequence binds V. cholerae; and a pharmaceutically acceptable carrier,wherein said composition is capable of binding the microorganism so thatit is eliminated from the gastrointestinal tract.

In yet a still further aspect, the invention provides a method to bindand remove cholera toxin and/or V. cholerae organisms from a samplesuspected of containing said toxin or organism, which method comprisescontacting said sample with an oligosaccharide sequence covalentlyattached to a solid, inert support through a non-peptidyl compatiblelinker arm, wherein said oligosaccharide sequence binds cholera toxinand/or V. cholerae organisms, under conditions wherein said choleratoxin and/or V. cholerae organism is absorbed to said support; andseparating the support containing the absorbed toxin and/or organismfrom the sample.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 demonstrates the neutralization of purified cholera toxincytotonic activity using a panel of SYNSORBs containing variousoligosaccharide sequences. Several SYNSORBs were found to effectivelyneutralize cholera toxin activity.

FIG. 2 illustrates the concentration dependent neutralization of choleratoxin activity using SYNSORB 16, 19, 41, 72, 75 and 88. All theseSYNSORBs can effectively neutralize more than about 75% of cholera toxinactivity at a concentration of 20 mg/ml.

FIG. 3 demonstrates the neutralization of cholera toxin and choleracytotoxin activity produced by O139 V. cholerae using SYNSORB 16, 41,72, 75 and 88 at a concentration of 20 mg/ml. Several SYNSORBs wereeffective at neutralizing both activities.

FIG. 4 demonstrates the neutralization of cholera toxin and choleracytotoxin activity produced by O1 (E1 Tor biotype) V. cholerae usingSYNSORB 16, 41, 72, 75 and 88 at a concentration of 20 mg/ml. SeveralSYNSORBs were effective at neutralizing both activities.

FIG. 5 illustrates the effectiveness of SYNSORBs 16 and 75 at reducingcholera toxin-mediated fluid secretion in rabbit intestinal loops.SYNSORB 75 utilized at a dose of 0.5 g/kg significantly reduced fluidsecretion in rabbit intestinal loops that had been treated with purifiedcholera toxin.

FIG. 6 illustrates the effectiveness of SYNSORBs 16 and 75 at reducingcholera toxin-mediated mannitol permeability in rabbit intestinal loops.SYNSORB 75 utilized at a dose of 0.1 g/kg and SYNSORB 16 at a dose of0.5 g/kg significantly reduced intestinal permeability in rabbitintestinal loops that had been treated with purified cholera toxin.

FIG. 7 demonstrates the effectiveness of SYNSORB in binding O1 V.cholerae (classical). The results show that classical biotypes of V.cholerae bind to the surface of SYNSORBs 1, 41, 57 and 90.

FIG. 8 demonstrates the effectiveness of SYNSORB in binding O1 V.cholerae (E1 Tor). The results show that E1 Tor biotypes of V. choleraebind to the surface of SYNSORBs 1, 5, 57 and 72.

FIG. 9 demonstrates the effectiveness of SYNSORB in binding O139 V.cholerae. The results show that O139 serotypes of V. cholerae bind toSYNSORBs 2, 5, 57 and 90.

DETAILED DESCRIPTION OF THE INVENTION

A. Definitions

As used herein the following terms have the following meanings:

The term "cholera" refers to an acute epidemic infectious disease causedby Vibrio cholerae, wherein a soluble toxin elaborated in the intestinaltract by the Vibrio alters the permeability of the mucosa, causing aprofuse watery diarrhea, extreme loss of fluid and electrolytes, and astate of dehydration and circulatory collapse, but no gross morphologicchange in the intestinal mucosa.

The term "biocompatible" refers to chemical inertness with respect tohuman tissues or body fluids. Biocompatible materials arenon-sensitizing.

The term "compatible linker arm" refers to a moiety which serves tospace the oligosaccharide structure from the biocompatible solid supportand which is biofunctional wherein one functional group is capable ofbinding to a reciprocal functional group of the support and the otherfunctional group is capable of binding to a reciprocal functional groupof the oligosaccharide structure. Compatible linker arms preferred inthe present invention are non-peptidyl spacer arms.

The term "solid support" refers to an inert, solid material to which theoligosaccharide sequences may be bound via a compatible linker arm.Where use is in vivo, the solid support will be biocompatible.

The term "SYNSORB" refers to synthetic 8-methoxycarbonyloctyloligosaccharide structures covalently coupled to Chromosorb P™ (ManvilleCorp., Denver, Colo.) [11], which is a derivatized silica particle.

The term "cholera toxin" refers to an enterotoxin of V. cholerae whichinitiates cholera and related conditions. This toxin has a lectin-likeactivity.

For purpose of this application, all sugars are referenced usingconventional three letter nomenclature. All sugars are assumed to be inthe D-form unless otherwise noted, except for fucose, which is in theL-form. Further all sugars are in the pyranose form.

B. Synthesis

Chemical methods for the synthesis of oligosaccharide structures can beaccomplished by methods known in the art. These materials are generallyassembled using suitably protected individual monosaccharides.

The specific methods employed are generally adapted and optimized foreach individual structure to be synthesized. In general, the chemicalsynthesis of all or part of the oligosaccharide glycosides firstinvolves formation of a glycosidic linkage on the anomeric carbon atomof the reducing sugar or monosaccharide. Specifically, an appropriatelyprotected form of a naturally occurring or of a chemically modifiedsaccharide structure (the glycosyl donor) is selectively modified at theanomeric center of the reducing unit so as to introduce a leaving groupcomprising halides, trichloroacetimidate, acetyl, thioglycoside, etc.The donor is then reacted under catalytic conditions well known in theart with an aglycon or an appropriate form of a carbohydrate acceptorwhich possesses one free hydroxyl group at the position where theglycosidic linkage is to be established. A large variety of aglyconmoieties are known in the art and can be attached with the properconfiguration to the anomeric center of the reducing unit.

Appropriate use of compatible blocking groups, well known in the art ofcarbohydrate synthesis, will allow selective modification of thesynthesized structures or the further attachment of additional sugarunits or sugar blocks to the acceptor structures.

After formation of the glycosidic linkage, the saccharide glycoside canbe used to effect coupling of additional saccharide unit(s) orchemically modified at selected positions or, after conventionaldeprotection, used in an enzymatic synthesis. In general, chemicalcoupling of a naturally occurring or chemically modified saccharide unitto the saccharide glycoside is accomplished by employing establishedchemistry well documented in the literature [12-28].

The solid supports to which the oligosaccharide structures of thepresent invention are bound may be in the form of sheets or particles. Alarge variety of biocompatible solid support materials are known in theart. Examples thereof are silica, synthetic silicates such as porousglass, biogenic silicates such as diatomaceous earth,silicate-containing minerals such as kaolinite, and synthetic polymerssuch as polystyrene, polypropylene, and polysaccharides. Solid supportsmade of inorganic materials are preferred. Preferably the solid supportshave a particle size of from about 10 to 500 microns for in vivo use. Inparticular, particle sizes of 100 to 200 microns are preferred.

The oligosaccharide structure(s) is covalently bound or noncovalently(passively) adsorbed onto the solid support. The covalent bonding may bevia reaction between functional groups on the support and the compatiblelinker arm of the oligosaccharide structure. It has unexpectedly beenfound that attachment of the oligosaccharide structure to thebiocompatible solid support through a compatible linking arm provides aproduct which, notwithstanding the solid support, effectively removestoxin. Linking moieties that are used in indirect bonding are preferablyorganic bifunctional molecules of appropriate length (at least onecarbon atom) which serve simply to distance the oligosaccharidestructure from the surface of the solid support.

The compositions of this invention are preferably represented by theformula:

    (OLIGOSACCHARIDE-Y-R).sub.n -SOLID SUPPORT

where OLIGOSACCHARIDE represents an oligosaccharide group of at least 2sugar units which group binds to cholera toxin and/or V. cholerae, Y isoxygen, sulfur or nitrogen, R is an aglycon linking arm of at least 1carbon atom, SOLID SUPPORT is as defined above, and n is an integergreater than or equal to 1. Preferred aglycons are from 1 to about 10carbon atoms. Oligosaccharide sequences containing about 1 to 10saccharide units may be used. Sequences with about 1 to 3 saccharideunits are preferred. Preferably, n is an integer such that thecomposition contains about 0.25 to 2.50 micromoles oligosaccharide pergram of composition.

Numerous aglycon linking arms are known in the art. For example, alinking arm comprising a para-nitrophenyl group (i.e., --OC₆ H₄ pNO₂)has been disclosed [29]. At the appropriate time during synthesis, thenitro group is reduced to an amino group which can be protected asN-trifluoroacetamido. Prior to coupling to a support, thetrifluoroacetamido group is removed thereby unmasking the amino group.

A linking arm containing sulfur has been disclosed [30]. Specifically,the linking arm is derived from a 2-bromoethyl group which, in asubstitution reaction with thionucleophiles, has been shown to lead tolinking arms possessing a variety of terminal functional groups such as--OCH₂ CH₂ SCH₂ CO₂ CH₃ and --OCH₂ CH₂ SC₆ H₄ --pNH₂. These terminalfunctional groups permit reaction to complementary functional groups onthe solid support, thereby forming a covalent linkage to the solidsupport. Such reactions are well known in the art.

A 6-trifluoroacetamido-hexyl linking arm (--O--(CH₂)₆ --NHCOCF₃) hasbeen disclosed [31] in which the trifluoroacetamido protecting group canbe removed, unmasking the primary amino group used for coupling.

Other exemplifications of known linking arms include the7-methoxycarbonyl-3,6,dioxaheptyl linking arm [32](--OCH₂ --CH₂)₂ OCH₂CO₂ CH₃); the 2-(4-methoxycarbonylbutancarboxamido)ethyl [33] (--OCH₂CH₂ NHC(O)(CH₂)₄ CO₂ CH₃); the allyl linking arm [34] (--OCH₂ CH═CH₂)which, by radical co-polymerization with an appropriate monomer, leadsto co-polymers; other allyl linking arms [35] are known (--O(CH₂ CH₂ O)₂CH₂ CH═CH₂). Additionally, allyl linking arms can be derivatized in thepresence of 2-aminoethanethiol [36] to provide for a linking arm --OCH₂CH₂ CH₂ SCH₂ CH₂ NH₂. Other suitable linking arms have also beendisclosed [12-14, 16, 17].

The particular linking employed to covalently attach the oligosaccharidegroup to the solid support is not critical.

Preferably, the aglycon linking arm is a hydrophobic group and mostpreferably, the aglycon linking arm is a hydrophobic group selected fromthe group consisting of --(CH₂)₈ C(O)--, --(CH₂)₅ OCH₂ CH₂ CH₂ -- and--(CH₂)₈ CH₂ O--.

We have found that synthetic oligosaccharide sequences covalentlyattached to a biocompatible solid support, e.g., Chromosorb P™ (SYNSORB)may be used to bind cholera toxin and/or V. cholerae. These compositionsare useful to treat cholera and associated conditions. SYNSORB isparticularly preferred for these compositions because it is non-toxicand resistant to mechanical and chemical deposition. In studies usingrats (a widely accepted model for preclinical studies, since they arepredictive of human response), SYNSORBs have been found to passunaffected through the rat gastrointestinal tract. They were found to beeliminated completely and rapidly (99% eliminated in 72 hours) followingoral administration.

Additionally, the high density of oligosaccharide moieties on SYNSORB isparticularly useful for binding cholera toxin, since the toxin isthought to possess multiple oligosaccharide binding sites [2]. The highdensity of oligosaccharide ligands on SYNSORB is also useful for bindinglarge numbers of V. cholerae.

Non-peptidyl linking arms are preferred for use as the compatiblelinking arms of the present invention. The use of glycopeptides is notdesirable because glycopeptides contain several, often different,oligosaccharides linked to the same protein. Glycopeptides are alsodifficult to obtain in large amounts and require expensive and tediouspurification. Likewise, the use of BSA or HSA conjugates is notdesirable, for example, due to questionable stability in thegastrointestinal tract when given orally.

Covalent attachment of an oligosaccharide group containing a choleratoxin or V. cholerae binding unit through a non-peptidyl spacer arm toan inert solid support permits efficient binding and removal of choleratoxin and/or microorganism from a sample to be analyzed for the presenceof cholera toxin and/or organism or from the intestine of a patientsuffering from cholera. When the oligosaccharide is synthesized withthis compatible linker arm attached (in non-derivatized form), highlypure compositions may be achieved which can be coupled to various solidsupports.

C. Pharmaceutical Compositions

The methods of this invention are achieved by using pharmaceuticalcompositions comprising one or more oligosaccharide structures whichbind cholera toxin and/or V. cholerae attached to a solid support.

When used for oral administration, which is preferred, thesecompositions may be formulated in a variety of ways. It will preferablybe in liquid or semisolid form. Compositions including a liquidpharmaceutically inert carrier such as water may be considered for oraladministration. Other pharmaceutically compatible liquids or semisolids,may also be used. The use of such liquids and semisolids is well knownto those of skill in the art.

Compositions which may be mixed with semisolid foods such as applesauce,ice cream or pudding may also be preferred. Formulations, such asSYNSORBs, which do not have a disagreeable taste or aftertaste arepreferred. A nasogastric tube may also be used to deliver thecompositions directly into the stomach.

Solid compositions may also be used, and may optionally and convenientlybe used in formulations containing a pharmaceutically inert carrier,including conventional solid carriers such as lactose, starch, dextrinor magnesium stearate, which are conveniently presented in tablet orcapsule form. The SYNSORB itself may also be used without the additionof inert pharmaceutical carriers, particularly for use in capsule form.

Doses are selected to provide neutralization and elimination of choleratoxin and/or elimination of V. cholerae found in the gut of the affectedpatient. Preferred doses are from about 0.25 to 1.25 micromoles ofoligosaccharide/kg body weight/day, more preferably about 0.5 to 1.0micromoles of oligosaccharide/kg body weight/day. Using SYNSORBcompositions, this means about 0.5 to 1.0 gram SYNSORB/kg bodyweight/day, which gives a concentration of SYNSORB in the gut of about20 mg/ml. Administration is expected to be 3 or 4 times daily, for aperiod of one week or until clinical symptoms are resolved. The doselevel and schedule of administration may vary depending on theparticular oligosaccharide structure used and such factors as the ageand condition of the subject. Optimal time for complete removal ofcholera toxin activity was found to be about 1 hour at 37° C., using aconcentration of SYNSORB of 20 mg in 1 ml sample. Similar conditions canbe used to effectively bind and remove V. cholerae from the gut.

Administration of the oligosaccharide-containing compositions of thepresent invention during a period of up to seven days will be useful intreating cholera and associated conditions. Also, prophylacticadministration will be useful to prevent colorization of the gut by V.cholerae and subsequent development of the disease.

As discussed previously, oral administration is preferred, butformulations may also be considered for other means of administrationsuch as per rectum. The usefulness of these formulations may depend onthe particular composition used and the particular subject receiving thetreatment. These formulations may contain a liquid carrier that may beoily, aqueous, emulsified or contain certain solvents suitable to themode of administration.

Compositions may be formulated in unit dose form, or in multiple orsubunit doses. For the expected doses set forth previously, orallyadministered liquid compositions should preferably contain about 1micromole oligosaccharide/ml.

D. Methodology

We have found that V. cholerae toxin may be neutralized by certainoligosaccharide sequences which bind the toxin. In particular, syntheticoligosaccharides covalently attached to solid supports via non-peptidylcompatible linker arms have been found to neutralize cholera toxineffectively. Examples of such compositions are certain SYNSORBs, whichbind and neutralize cholera toxin activity.

We have also found that V. cholerae bind to certain oligosaccharidesequences that are covalently attached to solid supports vianon-peptidyl compatible linker arms. Examples of such compositions arecertain SYNSORBs, which bind V. cholerae, thereby preventing theorganism from attaching to its host cell receptor in the intestinaltract before it is eliminated.

We have tested the ability of several oligosaccharide sequences attachedto Chromosorb P via an 8-methoxylcarbonyloctyl (MCO) spacer arm toneutralize cholera toxin and bind V. cholerae. The structures tested,also referred to as SYNSORBs, are presented in Table 1. As shown inFIGS. 1-4, the SYNSORBs tested varied in their ability to neutralize atleast about 50% of the cholera toxin activity. FIGS. 7-9 demonstrate theability of SYNSORB to bind V. cholerae.

The oligosaccharide sequences attached to solid supports useful in thepresent invention include those which bind cholera toxin. The bindingaffinity of an oligosaccharide to cholera toxin is readily detectable bya simple in vitro test, as for example, set forth in Example 1 below.For the purposes of this invention, oligosaccharide sequences attachedto solid supports which bind cholera toxin means those compositionswhich reduce endpoint titers from cytotonic activity in Chinese HamsterOvary (CHO) cell assays by at least 50%, using the assay set forth inthe Examples section.

Other oligosaccharide sequences attached to solid supports useful in thepresent invention are those which can bind V. cholerae significantlybetter (p≦0.05, using appropriate standard statistical methods, such asthe Wilcoxon or Student's T-test) than a control support that does notcontain any attached oligosaccharide sequences (e.g., Chromosorb P). Thebinding affinity of an oligosaccharide for V. cholerae is determined asoutlined in Example 6 below.

The binding of shiga-like toxins (SLTs) and Clostridium difficile toxinA to chemically synthesized oligosaccharide sequences has been studied[37-41].

SLTs are a group of cytotoxins which are made up of two parts: an Asubunit and a B oligomer. The B oligomer is the binding portion of thetoxin that allows it to bind to host cell receptors. The SLT toxins bindto glycolipid receptors containing the αGal(1-4)βGal determinant. The Asubunit has an enzymatic activity (N-glycosidase) that depurinates 28Sribosomal RNA in mammalian cells. This enzymatic activity abolishes theability of the toxin-infected cell to perform protein synthesis.

The site for SLT action is endothelial cells found in the kidneys andmesenteric vasculature, and SLTs may cause damage that can result inrenal failure and hemoglobin in the urine. SLTs are the causative agentin the hemolytic-uremic syndrome. SLTs may also be partially involved inthe pathogenesis of hemorrhagic colitis (bloody diarrhea).

Clostridium difficile toxin A is an enterotoxin that induces fluidsecretion, mucosal damage and intestinal inflammation. It serves as achemoattractant for human neutrophils. Toxin A is a single protein. Itcauses activation and results in the release of cytokines in monocytes.These inflammatory effects may play an important role in inducing thecolonic inflammation seen in pseudomembranous colitis.

Toxin A appears to bind to a glycoprotein receptor, the structure ofwhich has yet to be determined. The mechanism of action is not totallyunderstood, but toxin A is thought to enter cells via receptor-mediatedendocytosis and affect the actin cytoskeleton of the cell. The toxin Areceptor is thought to be linked to a guanine regulatory protein. ToxinA is the first step in the production of CDAD and PMC.

In contrast, cholera toxin is an AB₅ hexameric protein with fiveidentical B subunits and one A subunit. The B-pentamer recognizes andbinds to the cells of the intestine through a glycolipid receptor(ganglioside GM1). The A subunit, which is enzymatically active, is thentransported to the interior of the cell, where it causes elevated levelsof cyclic AMP, leading to the massive loss of fluids which characterizescholera and related conditions.

Previous studies defining the oligosaccharide binding specificity ofcholera toxin have identified several structural requirements for toxinbinding [1,5-10]. The major structural requirement for cholera toxinbinding is βGal(1-3)βGalNAc(1-4)[αNeuAc(2-3)]βGal [7]. Cholera toxin hasalso been shown to bind to galactose affinity columns, indicating thatterminal galactose sugars are important for toxin binding [8]. Theimportance of terminal galactose sugars is also confirmed in reducedbinding of cholera toxin to the ganglioside GM2(βGalNAc(1-4)[αNeuAc(2-3)]βGal(1-4)βGlc-ceramide) [6]. Sialic acid playsa major role in cholera toxin binding [1,5]. Removal of sialic acid fromGM1 to form asialo GM1 (βGal(1-3)βGalNAc(1-4)βGal(1-4)βGlc-ceramidedramatically reduces cholera toxin binding [6]. The SYNSORBs chosen fortoxin neutralization studies include carbohydrates that incorporateselected segments of the GM1 oligosaccharide structure. Other additionalSYNSORBs selected for binding studies contain oligosaccharide sequencesthat represent analogs of selected sequences in the GM1 gangliosidestructure. Oligosaccharide structures comprising a terminalβGal(1-3)βGal(1-4)βGal(1) moiety are also useful in the presentinvention.

The amount of cholera toxin adsorption to SYNSORB was determined byassaying supernatants for percent of toxin activity remaining relativeto controls without any added SYNSORB. Results are shown in FIGS. 1 and2. SYNSORBs 16, 19, 41, 72, 75 and 88 were found to effectively removecholera toxin activity. Four of these SYNSORBs (41, 72, 75 and 88)contained oligosaccharide sequences not previously shown to bind choleratoxin.

Thus, we have found that the ability to neutralize cholera toxin isdirectly related to the oligosaccharide sequences attached to the inertsupport. The results in FIGS. 1 and 2 show the importance of theβGal(1-3)βGalNAc linkage for high affinity toxin binding. In addition,we have found that oligosaccharide sequences which possessβGal(1-3)βGalNAc(1-4)βGal and αNeuAc(2-3)βGal show high affinity toxinbinding. We have further found that cholera toxin binds oligosaccharidesequences having βGal(1-3)βGal linkage. This structure represents ananalog of the βGal(1-3)βGalNAc sequence found in the GM1 structure.

The results presented in FIGS. 1 and 2 show percent toxin activityremaining. These results were obtained in tissue culture assays usingChinese hamster ovary (CHO) cells that showed a reduction in endpointdilution relative to controls when SYNSORB was added to purified choleratoxin.

Several different oligosaccharide sequences attached to solid supportsvia compatible linker arms have been found to have the ability toneutralize cholera toxin activity. These sequences, and others that alsobind cholera toxin, may be used to treat cholera and related conditions.Optimal time for complete removal of cholera toxin activity was found tobe about 1 hour at 37° C., using a concentration of SYNSORB of 20 mg in1 ml sample. Since each gram of SYNSORB contains approximately 0.25 to1.0 micromoles oligosaccharide, the total amount of oligosaccharide tobe given in a daily dose would range from 7.5 to 30 micromoles, using agut volume of four liters.

The utility of oligosaccharide sequences attached to a solid support viaa compatible linker arm to treat cholera was also demonstrated by theability of SYNSORB compositions to neutralize cholera toxin in an invivo animal model using rabbits. The results in FIGS. 5 and 6 and Table3 show that SYNSORB 75 can effectively reduce cholera toxin-mediatedfluid secretion and mannitol permeability in ligated rabbit intestinalloops. Further, the conditions used in the rabbit model best approximatethe actual conditions found in the human intestine.

Treatment of cholera or related conditions may be accomplished by oraladministration of compositions containing oligosaccharide sequencescovalently bound to a solid support via a compatible linker arm (e.g.SYNSORBs). For example, the SYNSORB has been found to pass through thestomach of rats intact. It then contacts the cholera toxin in theintestinal tract. Subsequent elimination of the intact SYNSORB withcholera toxin bound to it results in elimination of cholera toxin fromthe patient.

The primary virulence factor responsible for attachment of V. choleraeto epithelial cells in the intestine is the toxin coregulated pili. Thehost cell receptors used for the attachment process have not beendetermined, but there is indirect evidence that suggests that attachmentmay be mediated by blood group oligosaccharide sequences found onepithelial cells. The SYNSORBs chosen (Table 1) for bacterial attachmentstudies include carbohydrates related to the A, B and O blood groupstructures. Additional SYNSORBs chosen contain oligosaccharide sequencesthat were shown to bind to cholera toxin.

The amount of V. cholerae binding to the surface of SYNSORB wasdetermined by plating suspensions of SYNSORB that had been incubatedwith a culture of either O1 (Classical and E1 Tor) or O139 V. cholerae(1×10⁵ colony forming units (CFU)/ml). Control incubations were donewith V. cholerae and Chromosorb P, which does not have any attachedoligosaccharide sequences. The results in FIGS. 7-9 show that SYNSORBs1, 2, 5, 57, 72 and 90 bind one or more serotypes of V. cholerae. Allsix of these SYNSORBs contain oligosaccharide sequences that have notbeen previously shown to bind V. cholerae. These results also confirmepidemiological evidence that suggests a relationship between bloodgroup and an individual's susceptibility to cholera.

Thus, we have found that the ability to bind V. cholerae is directlyrelated to the oligosaccharide sequences attached to the inert support.The results in FIGS. 7-9 show the importance of theαGalNAc(1-3)[αFuc(1-2)βGal (Blood group A), αGal(1-3)[αFuc(1-2)βGal(Blood group B) and αFuc(1-2)βGal(1-4)βGlcNAc (H(O) blood group)linkages for V. cholerae binding. In addition, we have found thatoligosaccharide sequences which possess βGalNAc(1-4)βGal andβGal(1-3)βGal can also effectively bind V. cholerae. Accordingly,oligosaccharide sequences comprising βGal(1-4)βGal(2) will be useful inthe methods and composition of the present invention.

Treatment of cholera or related conditions may be accomplished by oraladministration of compositions containing oligosaccharide sequencescovalently bound to a solid support via a compatible linker arm (e.g.SYNSORBs). For example, the SYNSORB has been found to pass through thestomach of rats intact. It then contacts the organism V. cholerae in theintestinal tract. Subsequent elimination of the intact SYNSORB with V.cholerae bound to it results in elimination of the organism from thepatient.

Another aspect of the invention is the rapid efficient binding ofphysiological concentrations of cholera toxin or V. cholerae present inbiological samples, thus permitting assay of the presence and/orquantity of cholera toxin or organism in these samples. Typically, thebiological sample will be a stool sample. The sample may be extractedand prepared using standard extraction techniques. The sample or extractis then contacted with the toxin or organism binding oligosaccharidesequences covalently bound to solid supports via a compatible linker armunder conditions where any cholera toxin or V. cholerae in the sample isabsorbed.

Cholera toxin or V. cholerae may be measured directly on the surface ofthe oligosaccharide-containing support using any suitable detectionsystem. For example, radioactive, biotinylated or fluorescently labelledmonoclonal or polyclonal antibodies specific for cholera toxin may beused to determine the amount of cholera toxin bound to the support. Awide variety of protocols for detection of formation of specific bindingcomplexes analogous to standard immunoassay techniques is well known inthe art.

A panel of SYNSORBs (Table 1) was screened for the ability to neutralizepurified CT activity. The results in FIG. 1 show that SYNSORBs 16, 19,41, 72 75 and 88 removed 80%, 80%, 80%, 96%, 96% and 80% (n=2)respectively. The SYNSORBs that bound to CT with higher affinities fitvery well with data obtained from X-ray crystallographic studies whichshowed that the terminal disaccharide sequence (βGal(1-3)βGalNAc) aswell as the sialic acid sugar from the GM1 structure played major rolesin the interaction between toxin and carbohydrate [2]. The results fromFIG. 1 also showed that Chromosorb P did not appear to bind to CT.

Variable amounts of each SYNSORB were incubated with purified CT inorder to determine optimal binding conditions. The results fromneutralization experiments (FIG. 2) showed that SYNSORB used at aconcentration of 20 mg/ml should be effective at neutralizing CTactivity.

To determine whether the optimized conditions were effective atadsorbing CT activity from O1 serotypes of V. cholerae. Crude culturesupernatants from Classical and E1 Tor biotypes of V. cholerae wereincubated with SYNSORBs 16, 41, 72, 75 and 88. The results fromneutralization experiments with a classical biotype of O1 V. choleraeindicted that CT activity was reduced by 94±3%, 90±0%, 77±0%, 97±0% and97±0% (n=4) respectively for each of the SYNSORBs listed above. Usingtwo culture supernatants from E1 Tor biotypes of O1 V. cholerae (NIH V86and 95-0031), SYNSORBs 16, 41, 72, 75 and 88 reduced CT activity by81±0%, 75±0%, 89±0%, 81±0% and 75±0% (n=2) and 50±0%, 75±0%, 88±0%,66±0% and 94±0% (n=2) respectively.

Preliminary CT neutralization experiments with four O139 V. choleraeclinical isolates obtained from Dr. W. Johnson, LCDC, Ottawa revealedthe presence of a cytotoxic activity that is not found with theclassical O1 serotypes of V. cholerae. Two tissue culture assays areuseful for detecting CT activity. The classical CT assay involvesexposing Chinese hamster ovary (CHO) cells to solutions containing toxinand determining the cytotonic (cell elongation) end point after 24hours. The second involves HT 29 cells which produce large pleomorphicvacuoles when exposed to CT. Culture supernatants from O139 clinicalisolates had the ability to rapidly kill CHO cells (100% death in lessthan 24 hours) and induced vacuolization in HT 29 cells (Table 2). Theresults from preliminary experiments indicate some differences betweenthe O139 culture supernatants and purified CT.

To further explore the differences, neutralization experiments were donewith anti-CT antiserum. Dilutions of purified CT and O139 culturesupernatants were combined with anti-CT serum and incubated for 30minutes prior to adding the toxin dilutions to CHO and HT 29 cells.After incubating with toxin for 24 hours, the results indicated that thecytotoxic activity observed with CHO cells was not neutralized by theanti-CT antiserum. Antibody neutralization experiments using HT 29 cellsrevealed that the antiserum effectively reduced the formation ofvacuoles, suggesting the presence of CT in the culture supernatants.Control assays using purified CT showed good neutralization in both theCHO and HT 29 cells.

The data obtained from the neutralization assays suggest that two toxinactivities are produced by O139 strains. One of the activities is CT,which causes vacuolization in HT 29 cells. The second activity, acholera cytotoxin (CC) that kills CHO cells. Additional evidence tosupport the presence of CC in O139 culture supernatants was obtained byincubating toxin containing solutions with Vero cells which have beenshown to be resistant towards the effects of CT. Incubating O139 culturesupernatants with Vero cells resulted in rapid death of the cellsconfirming the presence an additional cytotoxic activity.

E1 Tor biotypes of V. cholerae are known to possess an additionalcytotoxic/hemolytic activity that is similar to CC produced by O139serotypes.

Preliminary neutralization studies with SYNSORBs 16, 41, 72, 75 and 88have shown that SYNSORB has the ability to adsorb CC and CT from culturesupernatants. The extent of CC neutralization was determined bycomparing the cytotoxic end points of SYNSORB treated culturesupernatants with untreated control samples using CHO cells. CTneutralization experiments were done in a similar manner except that HT29 cells were used to assess toxin levels. The results in FIG. 3 showthat SYNSORBs 16, 41, 72, 75 and 88 had the ability to neutralizegreater than 50% of CT activity in most cases. The results also showthat the cytotoxic activity produced by O139 serotypes and O1 E1 Torbiotypes may utilize oligosaccharide receptors similar to those used byCT for interacting with host cells. The ability of SYNSORB to neutralizeCT activity from O139 V. cholerae strains was somewhat reduced whencompared to the results obtained with the O1 serotype. The reducedaffinities for the various SYNSORBs may be due to slight differencesbetween the two CT activities.

E. Examples

The following methods were used to perform the studies in the Examplesthat follow.

Purified cholera toxin was obtained from Sigma Chemicals.

Preparation of Vibrio cholerae Culture Supernatants V. cholerae O1classical as well as E1 Tor biotypes were cultured in AKI media (15 gpeptone (Difco), 5 g NaCl, 4 g yeast extract (Difco) per liter water,adjusted to pH 7 with sodium bicarbonate) at 37° C. while clinicalisolates of V. cholerae O139 were grown in Syncase media (20 g casaminoacids (Difco), 8.7 g K₂ HPO₄, 6 g yeast extract (Difco), 25 g NaCl perliter water). Overnight cultures of V. cholerae were centrifuged at5,000×g for 30 min. to sediment the bacteria. The supernatants werecarefully removed and utilized in toxin neutralization studies.

Assay of Cholera Toxin Activity Using Tissue Culture Cells

The cytotonic activity of cholera toxin (CT) can be measured by the useof Chinese hamster ovary (CHO) cells that are maintained in Hams F12media supplemented with 10% fetal bovine serum (FBS) in an atmosphere of5% CO₂ at 37° C. CT samples to be tested were diluted 1:5 in Hams mediaand filter sterilized through 0.22 micron syringe filters. Samples to betested were serial 5-fold diluted in media and 100 μL of each dilutionwas added to wells with confluent monolayers of CHO cells and incubatedfor 24 h at 37° C. in 5% CO₂. Each sample was analyzed two times.Cytotonic effects were readily visible after 24 h incubation bycomparing wells with controls that do not contain toxin. After 24 h, thecells were fixed with 95% methanol and stained with Geimsa stain.

CT-containing samples from neutralization experiments were treated in ananalogous fashion except that the percent neutralization was determinedby comparing the endpoint dilutions of samples with and without SYNSORB.

Another cell line used to measure the effects of CT are human colonicadenocarcinoma HT 29 cells which are grown in the presence of 17 mMglucose using Dulbecco's Modified Eagles Medium (DMEM) plus 10% fetalbovine serum. CT containing solutions were serial 3 or 5-fold diluted inmedia and added to wells containing HT 29 cells. Pleomorphic vacuoleformation was readily visible after 24 h incubation by comparing samplewells with controls that did not contain any toxin.

Kidney cells from the African green monkey (VERO) were used as a controlcell line since they are resistant towards the effects of CT. VERO cellswere maintained in minimum essential medium (MEM) containing 3% FBS.

Assay of Cholera Cytotoxin Activity Using Tissue Culture Cells

Cholera cytotoxin activity was measured in an identical manner asdescribed above using either CHO or Vero cells.

The following examples are offered to illustrate this invention and arenot meant to be construed in any way as limiting the scope of thisinvention.

EXAMPLE 1 Screening of Oligosaccharide-Containing Solid Supports for theAbility to Neutralize Cholera Toxin Activity

A solution containing purified CT (2 μg in 1 ml PBS) was added tovarious SYNSORBs (amounts ranging from 20.0 to 22.5 mg) containingdifferent oligosaccharide sequences in 1.5 ml microcentrifuge tubes andincubated at room temperature for 1 h on a end-over-end rotator. Afterincubation, the SYNSORB was allowed to settle to the bottom of the tubesand the supernatants were carefully removed. Serial five-fold dilutionsof the supernatants were prepared and the cytotonic endpoint determinedas described above. The extent of reduction in the endpoint in thepresence of SYNSORB was determined by comparing with controls in whichSYNSORB was not added. An additional control utilized was Chromosorbwhich is void of any carbohydrate ligand.

Results are shown in FIG. 1, and demonstrate that severaloligosaccharide structures were found to effectively neutralize choleratoxin activity.

EXAMPLE 2 Concentration Dependent Neutralization of Cholera ToxinActivity Using SYNSORB 16, 19, 41, 72, 75 and 88

The amount of SYNSORBs 16, 19, 41, 72, 75 and 88 required for maximalcholera toxin neutralization was determined by adding 1 ml of a purifiedcholera toxin solution containing 2 μg cholera toxin (CT) to pre-weighedamounts of each SYNSORB in 1.5 ml microcentrifuge tubes. SYNSORB sampleswere tested using 10, 20 and 40 mg amounts. Samples were incubated for 1hour at 37° C. on an end-over-end rotator. Control samples containingonly cholera toxin solution were also tested.

The amount of neutralization in each sample was determined by comparingthe endpoint titers of CHO cell assays from samples with and withoutSYNSORB. The results, shown in FIG. 2, demonstrate that about 20 mg ofeach SYNSORB tested was able to neutralize at least 75% of the choleratoxin in 1 ml of cholera toxin solution.

EXAMPLE 3 Characterization of Cholera Cytotoxin Produced by O139 and O1(E1 Tor) Biotypes of V. cholerae

Anti-Cholera Toxin Neutralization Assays

Neutralization experiments were done with CHO or HT 29 tissue culturecells. Dilutions of culture supernatants of V. cholerae clinicalisolates were prepared and incubated with rabbit anti-CT antiserum(diluted 100 and 1000 times in each CT dilution) for 30 minutes at 37°C. Neutralization was determined by comparing end point dilution titersof supernatants that were treated with antiserum to titers of untreatedsamples. All experiments were done in duplicate.

The results in Table 2 show that the cytotoxic activity produced by O139clinical isolates was similar to the cytotoxin activity produced by O1E1 Tor biotypes of V. cholerae. The results in FIG. 4 also show that twotoxins are present in E1 Tor biotypes of V. cholerae.

EXAMPLE 4 Screening of Oligosaccharide-Containing Solid Supports for theAbility to Neutralize Cholera Toxin and Cholera Cytotoxin Activitiesfrom O139 and O1 E1 Tor Biotypes of V. cholerae

Crude culture supernatants from O139 and O1 E1 Tor biotypes of V.cholerae (1 ml) were added to various SYNSORBs (amounts ranging from20.0 to 22.5 mg) containing different oligosaccharide sequences in 1.5ml microcentrifuge tubes and incubated at room temperature for 1 h on aend-over-end rotator. After incubation, the SYNSORB was allowed tosettle to the bottom of the tubes and the supernatants were carefullyremoved. Serial three or five-fold dilutions of the supernatants wereprepared and the cytotonic or cytotoxic endpoints determined asdescribed above. The extent of reduction in the endpoint in the presenceof SYNSORB was determined by comparing with controls in which SYNSORBwas not added. An additional control utilized was Chromosorb which isvoid of any carbohydrate ligand.

Results are shown in FIGS. 3 and 4, and demonstrates the neutralizationof cholera cytotoxin and cholera toxin activity using SYNSORBs at aconcentration of 20 mg/ml. The results in FIGS. 3 and 4 indicate thatseveral oligosaccharide structures were found to effectively neutralizeboth toxin activities.

EXAMPLE 5 Determination of the Efficacy of SYNSORB in Reducing theEffects of Cholera Toxin in the Small Intestine of Rabbits

Specific pathogen free (SPF) male New Zealand white rabbits weighingapproximately 2 kg were used to assess the potential of SYNSORB toreduce the effects of CT in the ileum. Prior to surgery, each rabbit wasfasted but provided with water ad libitum. Rabbits were thenanesthetized by administering Isoflorane by means of a face mask. Usingsterile techniques, the abdomen was opened along the midsection and fourto six 10 cm long segments of ileum were ligated with umbilical tape toform loops. Each loop was separated by a 5 cm intestinal segment. Toeach loop was added 1.5 ml of a suspension of either SYNSORB 16 or 75 ata specified dose (0.1 g/kg or 0.5 g/kg) in 6 ml of 0.5% carboxymethylcellulose. Each intestinal loop of control animals received only 1.5 mlof 0.5% carboxymethyl cellulose.

Prior to addition of purified CT to the intestinal loops, ten μCi of ³H-mannitol (100 μL) was injected intravenously into the ear vein of eachrabbit by syringe. Each intestinal loop was then injected with either100 μL of a CT solution (25 μg) or phosphate buffered saline bytuberculin syringe. The intestinal loops were then replaced in theabdomen and the incision closed. Each rabbit was maintained under lightIsoflorane anesthesia for 4 hours, maintaining body temperature constantat 37° C. by placing each rabbit on a water circulating heating pad forthe duration of the experiment. At the termination of the incubationperiod, rabbits were then sacrificed by an intravenous injection ofpentobarbital. The ileal loops were removed, weighed and their lengthsmeasured. The loop contents were carefully removed and assayed for loopvolume as well as the amount of ³ H-mannitol in each loop.

The results from our studies (FIGS. 5 and 6, Table 3) show that SYNSORB75 was effective at minimizing CT activity in the intestines of rabbits.

EXAMPLE 6 Binding Experiments Using SYNSORB and Vibrio cholerae

Binding experiments were done by incubating approximately 10⁵ CFU of O1V. cholerae (Classical and E1 Tor biotypes) or O139 V. cholerae in 0.5ml of PBS with SYNSORBs 1, 2, 5, 57, 72 or 90 and Chromosorb P (20 mg)for 30 min. at room temperature. After extensive washing of the SYNSORBwith PBS (about 20 ml) to remove non adherent organisms, the SYNSORB wassuspended in 1 ml of 0.5% (w/v) carboxymethyl cellulose and twodilutions of the suspension were plated on nutrient agar plates. After24 h the plates were counted to determine the number of bound Vibrios.

The results in FIGS. 7-9 show that V. cholerae can effectively bind tothe surface of SYNSORB. The results also indicate that severaloligosaccharide structures were found to effectively serve as bindingsites for V. cholerae. The binding to SYNSORB is related to theoligosaccharide sequences found on SYNSORB since there is a significantdifference between organism binding to SYNSORB and Chromosorb P alone.The results in FIGS. 7-9 represent an average of at least 4determinations.

Modification of the above-described modes of carrying out variousembodiments of this invention will be apparent to those skilled in theart following the teachings of this invention as set forth herein. Theexamples described above are not limiting, but are merely exemplary ofthis invention, the scope of which is defined by the following claims.

                  TABLE 1                                                         ______________________________________                                        SYNSORBs Used in Cholera Toxin Neutralization Studies                         SYNSORB Structure Common    Oligosaccharide                                   Number  Number    Name      Structure*                                        ______________________________________                                        1       1         A         αGalNAc(1-3)βGal                                                   (1-2)                                                                         αFuc                                        2       2         B         αGal(1-3)βGal                                                      (1-2)                                                                         αFuc                                        5       3         H Type 2  βGal(1-4)βGlcNAc                                                    (1-2)                                                                         αFuc                                        16      4         lactose   βGal(1-4)βGlc                           19      5                   βGal                                         41      6         --        βGal(1-3)βGalNAc                        57      7         --        βGalNAc(1-4)βGal                        75      8         --        βGal(1-3)βGalNAc(1-4)βGal          88      9         --        αNeuAc(2-3)βGal                        72      10        --        βGal(1-3)βGal                           90      11        --        αGal(1-3)βGal(1-4)βGlc            ______________________________________                                         *All oligosaccharides are linked to Chromosorb P through a hydrophobic 8      carbon spacer arm. NeuAc is the abbreviation for sialic acid.            

                                      TABLE 2                                     __________________________________________________________________________    Effects of Culture Supernatants from V. cholerae O1 and O139 on Tissue        Culture Cells*                                                                            Anti-CT       Anti-CT                                                         Neutralization                                                                              Neutralization                                      Serotype                                                                            CHO Cells                                                                           (CHO Cells)                                                                          HT 29 Cells                                                                          (HT 29 Cells)                                                                        Vero Cells                                   __________________________________________________________________________    Purified CT                                                                         cytotonic                                                                           yes    vacuolization                                                                        yes    no effect                                    O1 (Inaba)                                                                          cytotonic                                                                           yes    vacuolization                                                                        yes    no effect                                    O139-93-302                                                                         cytotoxic                                                                           no     vacuolization                                                                        yes    cytotoxic                                    O139-93-329                                                                         cytotoxic                                                                           no     vacuolization                                                                        yes    cytotoxic                                    O139-93-520                                                                         cytotoxic                                                                           no     vacuolization                                                                        yes    cytotoxic                                    O139-93-695                                                                         cytotoxic                                                                           no     vacuolization                                                                        yes    cytotoxic                                    O1 El Tor                                                                           cytotoxic                                                                           no     vacuolization                                                                        yes    N.D.                                         NIH V86                                                                       O1 El Tor                                                                           cytotoxic                                                                           no     vacuolization                                                                        yes    N.D.                                         95-0031                                                                       __________________________________________________________________________     *Neutralization experiments were done with Chinese hamster ovary (CHO) or     human colonic adenocarcinoma (HT 29) tissue culture cells. Dilutions of       culture supernatants of V. cholerae clinical isolates were prepared and       incubated with antiCT for 30 minutes at 37° C. Neutralization was      determined by comparing end point dilution titres of supernatants that        were treated with antiserum with untreated samples. All experiments were      done in duplicate.                                                            N.D. not done.                                                           

                  TABLE 3                                                         ______________________________________                                        Neutralization of the Effects of CT in Ligated Rabbit Ileal Loops                                     p*   p+                                               ______________________________________                                                        Fluid Secretion                                                               (g/mm loop)                                                   Chlolera Toxin (n = 10)                                                                         0.019 ± 0.003                                                                          --     --                                       CT + SYNSORB 75, 0.5 g/kg (n = 7)                                                               0.006 ± 0.001                                                                          0.018  <0.001                                   CT + SYNSORB 75, 0.1 g/kg (n = 4)                                                               0.013 ± 0.001                                                                          0.465  0.380                                    CT + SYNSORB 16, 0.5 g/kg (n = 7)                                                               0.020 ± 0.003                                                                          0.735  0.828                                    CT + SYNSORB 16, 0.1 g/kg (n = 4)                                                               0.029 ± 0.007                                                                          1.000  0.692                                                    Fluid                                                                         Volume (ml)                                                   Chlolera Toxin (n = 10)                                                                         3.46 ± 0.57                                                                            --     --                                       CT + SYNSORB 75, 0.5 g/kg (n = 7)                                                               1.08 ± 0.38                                                                            0.018  <0.001                                   CT + SYNSORB 75, 0.1 g/kg (n = 4)                                                               2.18 ± 0.68                                                                            0.068  0.045                                    CT + SYNSORB 16, 0.5 g/kg (n = 7)                                                               3.26 ± 0.55                                                                            0.612  0.549                                    CT + SYNSORB 16, 0.1 g/kg (n = 4)                                                               3.70 ± 1.14                                                                            1.000  0.954                                                    .sup.3 H-Mannitol                                                             Permeability                                                                  (cpm loop)                                                    Chlolera Toxin (n = 10)                                                                         2821 ± 984                                                                             --     --                                       CT + SYNSORB 75, 0.5 g/kg (n = 7)                                                               234 ± 55 0.109  0.122                                    CT + SYNSORB 75, 0.1 g/kg (n = 4)                                                                0 ± 31  0.068  0.023                                    CT + SYNSORB 16, 0.5 g/kg (n = 7)                                                               1024 ± 355                                                                             0.068  0.029                                    CT + SYNSORB 16, 0.1 g/kg (n = 4)                                                               1529 ± 782                                                                             0.593  0.602                                    ______________________________________                                         *The P values shown indicate the significance of any differences between      the degree of either fluid secretion, fluid volume or mannitol                permeability in SYNSORB treated rabbit ileal loops and untreated ileal        loops. The P values were determined using the nonparametric Wilcoxon test     on SYSTAT computer software.                                                  +The P values were determined using the Students T test on SYSTAT compute     software.                                                                

What is claimed is:
 1. A method to treat cholera in a subject, whichmethod comprises administering to a subject in need of such treatment aneffective amount of a composition comprising an oligosaccharide sequencecovalently attached to a pharmaceutically acceptable solid, inertsupport through a non-peptidyl compatible linker arm, wherein saidoligosaccharide sequence when so bound to said solid, inert support iscapable of binding one or more serotypes of V. cholerae, and whereinsaid composition is capable of being eliminated from thegastrointestinal tract.
 2. The method of claim 1 wherein saidoligosaccharide sequence has from 1 to 3 saccharide units.
 3. The methodof claim 1 wherein said oligosaccharide sequence is selected from thegroup consisting of ##STR1##
 4. The method of claim 1 wherein saidlinker arm is --(CH₂)₈ C(O)--.
 5. A pharmaceutical composition useful intreating cholera, which composition comprises:a) an oligosaccharidesequence covalently attached to a pharmaceutically acceptable solid,inert support through a non-peptidyl compatible linker arm, wherein saidoligosaccharide sequence when so bound to said solid, inert support iscapable of binding one or more serotypes of V. cholerae; and b) apharmaceutically acceptable carrier, wherein said composition is capableof being eliminated from the gastrointestinal tract.
 6. The compositionof claim 5 wherein said oligosaccharide sequence has from 1 to 3saccharide units.
 7. The composition of claim 5 wherein saidoligosaccharide sequence is selected from the group consisting of##STR2##
 8. The composition of claim 5 wherein said linker arm is--(CH₂)₈ C(O)--.
 9. A method to bind and remove one or more serotypes ofV. cholerae from a sample suspected of containing said V. cholerae,which method comprises:a) contacting said sample with an oligosaccharidesequence covalently attached to a solid support through a non-peptidylcompatible linker arm, wherein said oligosaccharide sequence when sobound to said solid, inert support is capable of binding one or moreserotypes of V. cholerae, under conditions wherein said V. cholerae isabsorbed to said solid support; and b) separating the support containingthe absorbed V. cholerae from the sample.
 10. The method of claim 9wherein said oligosaccharide sequence has from 1 to 3 saccharide units.11. The method of claim 9 wherein said oligosaccharide sequence isselected from the group consisting of ##STR3##
 12. The method of claim 9wherein said linker arm is --(CH₂)₈ C(O)--.